Ganglioside associated recombinant antibodies and the use thereof in the diagnosis and treatment of tumors

ABSTRACT

The present invention is related with the obtaining of modified antibodies by means of the DNA recombinant technology from the murine monoclonal antibody P3 (MAb P3) produced by the hybridoma cell line deposited under Budapest Treaty with accession number ECACC 94113026 and from its anti-idiotype murine monoclonal antibody 1E10 (MAbai 1E10) produced by the hybridoma cell line with deposit number ECACC 97112901, with the objective of achieving monoclonal antibodies which preserve the biological function of specific binding to the antigen of the original antibodies, but being at the same time less immunogenic. The chimeric antibodies of the invention contain the variable domains of the murine immunoglobulin and the constant regions of the human immunoglobulin; and those humanized, besides containing the constant regions of the human immunoglobulins, they are modified in the region of the murine frameworks (FRs) and in particular in those zones that could be in an antigenic site for the T cells, so several positions of the FRS are human as well. These antibodies can be used in the diagnosis and therapy of different types of tumors. The present invention is also related with use of the antibodies for therapeutical and diagnostic purposes.

PRIOR RELATED APPLICATIONS

This patent application is a divisional patent application of U.S. Pat.No. 10,473,977, filed Nov. 17, 2003, which claims priority to PCT PatentApplication PCT/CU2002/00003, filed Apr. 8, 2002 and Cuban PatentApplication CU 84/2001, filed Apr. 6, 2001 and incorporates theseapplications in their entireties by reference thereto.

BACKGROUND

1. Field of the Invention

The present invention is related to the biotechnology field, inparticular with new recombinant antibodies obtained by geneticengineering, specifically with chimeric and humanized antibodiesobtained from the murine monoclonal antibody P3 (MAb P3) and itsanti-idiotype murine monoclonal antibody 1E10 (MAbai 1E10).

More specifically, the invention is related with antibodies that bind togangliosides containing N-glycolylated sialic acid, but not with theacetylated forms of the gangliosides neither with neuter glycolipids.Gangliosides containing N-glycolylated sialic acid are antigens widelyexpressed in breast cancer and melanomas. On the other hand, theanti-tumor effect of the MAbai 1E10 has also been demonstrated inexperimental models.

The present invention is also related with the pharmaceuticalcompositions that contain the previously described recombinantantibodies useful in the diagnosis and therapy of cancer, particularlybreast cancer and melanomas.

2. Discussion of the Prior Art

Gangliosides are glycosphingolipids that contain sialic acid and theyare present in the plasmatic membrane of cells of vertebrates (Stults etal. (1989):

Glycosphingolipids: structure, biological source and properties, MethodsEnzymology, 179:167-214). Some of these molecules have been reported inthe literature as antigens associated to tumors or tumor markers(Hakomori et al. (1991): Possible functions of tumor associatedcarbohydrate antigens, Curr. Opin. Immunol., 3: 646-653), for thatreason the use of anti-gangliosides antibodies has been described asuseful in the diagnosis and therapy of cancer (Hougton et al. (1985):Mouse monoclonal antibody IgG3 antibody detecting GD3 ganglioside: tophase I trial in patients with malignant melanoma, PNAS USA,82:1242-1246; Zhang et al. (1997): Selection of carbohydrate tumorantigens as targets for immune attack using immunohistochemistry. I.Focus on gangliosides, Int. J. Cancer, 73: 42-49).

Sialic acids more frequently expressed in animals are N-acetyl (NeuAc)and N-glycolyl (NeuGc)(Corfield et al. (1982): Occurrence of sialicacids, Cell. Biol. Monogr., 10: 5-50). NeuGc is not expressed in normalhuman and chickens tissues in general, but it is broadly distributed inother vertebrates (Leeden and Yu, (1976): Chemistry and analysis ofsialic acid. In: Biological Role of Sialic Acid. Rosemberg A andShengtrund C L (Eds). Plenum Press, New York, 1-48; Kawai et al. (1991):Quantitative determination of N-glycolylneuraminic acid expression inhuman cancerous tissues and avian lymphoma cell lines as a tumorassociated sialic acid by gas chromatography-mass spectrometry, CancerResearch, 51: 1242-1246). However, there are reports that show thatantibodies anti-NeuGc, recognize some human tumors and tumor cell lines(Higashi et al. (1988): Detection of gangliosides asN-glycolylneuraminic acid specific tumor-associated Hanganutziu-Deicherantigen in human retinoblastoma cells, Jpn. J. Cancer Res., 79: 952-956;Fukui et al. (1989): Detection of glycoproteins as tumor associatedHanganutziu-Deicher antigen in human gastric cancer cell line, NUGC4,Biochem. Biophys. Res. Commun., 160: 1149-1154). Increased levels of GM3(NeuGc) gangliosides have been found in human breast cancer (Marquina etal. (1996): Gangliosides expressed in human breast cancer, CancerResearch, 1996; 56: 5165-5171), this result makes attractive the use ofthis molecule as target for cancer therapy.

The monoclonal antibody (Mab) P3 produced by the cell line depositedwith accession number ECACC 94113026 (European Patent EP 0 657 471 B1),it is a murine monoclonal antibody with IgM isotype, that was obtainedwhen fusing murine splenocytes from a BALB/c mouse immunized withliposomes containing GM3(NeuGc) and tetanic toxoid, with the cell lineP3-X63-Ag8.653; which is a murine myeloma. This Mab P3 reacts stronglywith gangliosides containing N-glycolylated sialic acid but not with theacetylated forms of the gangliosides neither with the neuterglycolipids. It was demonstrated by immunocytochemical andimmunohistochemical studies carried out with cell lines and tissues frombenign and neoplasic tumors that the Mab P3 recognizes breast cancer(Vzquez et al. (1995): Generation of a murine monoclonal antibodyspecific for N-glycolylneuraminic acid-containing gangliosides that alsorecognizes sulfated glycolipids, Hybridoma, 14: 551-556) and melanoma.

The Mab P3 induced anti-idiotypic immune response (Ab2) in mice BALB/c(syngeneic model), even without adjuvant and carrier protein, (Vasquezet al. (1998): Syngeneic anti-idiotypic monoclonal antibodies to ananti-NeuGc-containing ganglioside monoclonal antibody, Hybridoma, 17:527-534). The role of the electronegative groups, sialic acid (forgangliosides) and SO₃—(for sulfatides), in the recognition properties ofthis antibody was suggested by immunochemical analysis (Moreno et al.(1998): Delineation of epitope recognized by an antibody specific forN-glycolylneuraminic acid-containing gangliosides, Glycobiology, 8:695-705).

The anti-idiotypic Mab 1E10 (Mabai 1E10) of subtype IgG1, was obtainedfrom a mouse BALB/c immunized with the Mab P3 coupled to KLH (U.S. Pat.No. 6,063,379, cell line deposited under accession number ECACC97112901). Mabai 1E10 recognized specifically the MAb P3 and it did notbind other IgM anti-ganglioside antibodies. Moreover, Mabai 1E10inhibited the specific binding of Mab P3 to the GM3(NeuGc) and to a cellline MDA-MB-435 derived from ductal breast carcinoma (positive for MabP3 binding). The MAbai 1E10 induced a strong immune response of Ab3antibodies when mice from syngeneic or alogenic models were immunized,these Ab3 antibodies didn't exhibit the same specificity as the Mab P3eve when they carry idiotopes similar to those carried by the Ab1antibody (Vzquez et al. (1998): Syngeneic anti-idiotypic monoclonalantibodies to an anti-NeuGc-containing ganglioside monoclonal antibody,Hybridoma, 17: 527-534). MAbai 1E10 induced a strong antitumor effect insyngeneic as well as alogenic mice. The growth of the mammary carcinomacell line F311 was significantly reduced by the repeated dose of theMAbai 1E10 coupled KLH in Freund's adjuvant, when BALB/c mice werevaccinated. Also the number of spontaneous lung metastasis was reducedafter the vaccination. The intravenous administration of the Mabai 1E10to C57BLU6 mice inoculated, 10 to 14 days after the intravenousinoculation of melanoma cells B16, caused a dramatic reduction of thenumber of lung metastases when compared with mice treated with anirrelevant IgG. These results suggest that more than one mechanism ofanti tumor effect is triggered (Vasquez et al. (2000): Anti tumorproperties of an anti-idiotypic monoclonal antibody in relation toN-glycolyl-containing gangliosides, Oncol. Rep., 7: 751-756, 2000).

Even when hybridoma technology has been developed during 15 years(Koehler y Milstein (1975): Continuous cultures of fused cells secretingantibody of predefined specificity, Nature, 256: 495497) and whenmonoclonal antibodies are still very useful in diagnosis as well asresearch they have not demonstrated their therapeutic effectiveness inhuman. It has been mainly due to their short half-life in blood and tothat murine effector functions fail for the human immune system, andalso for the human anti-mouse antibody immune response (HAMA response).

Otherwise, genetic engineering technology has revolutionized thepotential of the MAb utility, since manipulating immunoglobulin genes itis possible to obtain modified antibodies with reduced antigenicity, aswell as to improve its effector functions for the treatment or diagnosisof certain pathologies. Methods for reducing immunoglobulinimmunogenicity have as essential objective to diminish the differencesbetween a murine antibody and a human immunoglobulin, without alteringthe antigen recognition specificity (Morrison y Oi (1989): Geneticallyengineered antibody molecules, Adv Immunol., 44: 65-92).

Recently several methods have been developed to humanize murine or ratantibodies and, of this way, to reduce the xenogenic immune responseagainst foreign proteins when they are injected in humans. One of thefirst approach to reduce the antigenicity were the chimeric antibodies,in which the variable domains of the murine protein are inserted inconstant domains of human molecules, that exhibit the same specificitybut reduced immunogenicity compared to their murine counterparts, humaneffector functions are preserved by chimeric antibodies, (Morrison etal. (1984): Chimeric human antibody molecules: Mouse antigen-bindingdomains with human constant region domains, PNAS USA, 81: 6851-6855).Even when chimeric antibodies have the same specificity as the murinecounterpart, an immune response to the rodent variable regions isfrequently observed.

In an attempt to further reduce the immunogenicity of chimericantibodies, only the CDRs from the rodent monoclonal antibody have beengrafted onto human framework regions and this hybrid variable regionexpressed with human constant regions (Jones et al. (1986): Replacingthe complementary-determining regions in a human antibody with thosefrom a mouse, Nature 321: 522-524; Verhoeyen et al. (1988): Reshapinghuman antibodies: grafting an antilysozyme activity, Science 239,1534-1536). However, this approach has several shortcomings: frequentlythe resulting antibody has decreased affinity and a number of frameworkresidues must be backmutated to the corresponding murine ones to restorebinding (Rietchmann et al. (1988): Reshaping human antibodies fortherapy, Nature, 332: 323-327; Queen et al. (1989): A humanized antibodythat binds to the inteneukin 2 receptor, PNAS USA, 86: 10029-10033;Tempest et al. (1991): Reshaping a human monoclonal antibody to inhibithuman respiratory syncytial virus infection in vivo, Biotechnology, 9:266-272). In addition, persisting immunogenicity is frequently observedin the CDR-grafted antibodies.

Mateo and collaborators (U.S. Pat. No. 5,712,120) have described aprocedure for reducing immunogenicity of murine antibodies. According tothe method, the modifications are restricted to the variable domains andspecifically to the murine FRs of chimeric antibodies. Moreover, thereplacements are only carried out in those regions of the FRs that haveamphipatic sequences and therefore they are potential epitoposrecognized by T cells.

The method comprises judiciously replacement of few amino acid residues,located in the potential immunogenic epitopes by the correspondingresidues from the most homologous human sequence, the amino acids thatare mainly responsible for canonical structures and also the residues inthe immediate neighborhood of the CDRs or into the Vernier zone, must beretained.

The resulting antibody retains its antigen binding specificity and to beless immunogenic than either its murine or chimeric predecessor (Mateoet al. (2000): Removal of T cell epitopes from genetically engineeredantibodies: Production of modified immunoglobulins with reducedimmunogenicity, Hybridoma 19: 463-71), these properties increases theirtherapeutic utility. Using this new procedure only few mutations, and ofcourse less genetic manipulations, have to be done.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is related to recombinant antibodies, obtained bygenetic engineering technology. Specifically, the invention is relatedwith a chimeric antibody derived from the murine monoclonal antibody P3,produced by hybridoma cell line with deposit number ECACC 94113026. MABP3 recognizes an antigen expressed in breast tumor cells and melanomas.The MAb P3 is characterized by the following sequences of thehypervariable regions (CDRs) of the heavy and light chains:

Heavy Chain CDR1: RYSVH CDR2: MIWGGGSTDYNSALKS CDR3: SGVREGRAQAWFAYCadena Ligera CDR1: KASQDVSTAVA CDR2: SASYRYT CDR3: QQHYSTPWTPreferably, the FRs sequences of the heavy and light chain are thefollowing:

Heavy Chain FR1: QVQLKESGPGLVAPSQSLSITCTVSGFSLS FR2: WVRQPPGKGLEWLG FR3:RLSISKDNSKSQVFLKMNSLQTDDTAMYYCAR FR4: WGQGTLV Light Chain FR1:DIVMTQSHKFMSTSVGDRVSITC FR2: WYQQKPGQSPKLLIY FR3:GVPDRFTGSGSGTDFTFTISSVQAEDLAVYYC FR4: FGGGTKL

In a preferred embodiment, the chimeric antibody of the presentinvention, contains the constant region of heavy chain human IgG1 andthe constant region of light chain human Ck. In another aspect, thepresent invention is related with a humanized antibody derived from theMab P3 produced by the hybridoma cell line with deposit number ECACC94113026, characterized because it contains the constant region of humanheavy chain IgG1 and the constant region of human light chain human Ckand FRs regions of the light chain contains any of the following pointmutations:

Light Chain: Position 8: His by Pro Position 9: Lys by Ser Position 10:Phe by Ser Position 11: Met by Leu Position 13: Thr by Ala

In another aspect, the invention is related with a chimeric antibodyderived from the murine monoclonal antibody 1E10 produced by thehybridoma cell line with deposit number ECACC 97112901, and it is anantidiotype antibody, which recognizes the MAb P3. The MAbai 1E10 ischaracterized by the following sequences of the hypervariable regions(CDRs) of the heavy and light chains:

Heavy Chain CDR1: SYDIN CDR2: WIFPGDGSTKYNEKFKG CDR3: EDYYDNSYYFDY LightChain CDR1: RASQDISNYLN CDR2: YTSRLHSG CDR3: QQGNTLPWT

Preferably, the FRs sequences of the heavy and light chain are thefollowing:

Heavy Chain FR1: QVQLQQSGAELVKPGASVKLSCKASGYTFT FR2: WVRQRPEQGLEWIG FR3:KATLTTDKSSSTAYMQLSRLTSEDSAVYFCAR FR4: WGQGTTLTV Light Chain FR1:DIQMTQTTSSLSASLGDRVTISC FR2: WYQQKPDGTVKLLIY FR3:VPSRFSGSGSGTDYSLTISNLEQEDIATYFC FR4: FGGGTKLESK

In a preferred embodiment, the chimeric antibody of the presentinvention, contains the constant region of heavy chain human IgG1 andthe constant region of light chain human Ck.

In another aspect, the present invention is related with a humanizedantibody derived from the Mab 1E10 produced by the hybridoma cell linewith deposit number ECACC 97112901, characterized in that it containsthe constant region of human heavy chain IgG1 and the constant region ofhuman light chain Ck and FRs regions of the heavy and light chaincontains any of the following point mutations:

Light Chain: Position 7: Thr by Ser Position 8: Thr by Pro Position 15:Leu by Val Heavy Chain: Position 5: Gln by Val Position 40: Arg by AlaPosition 42: Glu by Gly Position 87 (83 according Kabat's numbering):Thr by Arg

In another aspect, the present invention is related with the cell linesthat express the described chimeric and humanized antibodies;additionally the invention is related with pharmaceutical compositionscomprising the described antibodies.

Preferably it is related with pharmaceutical compositions for thetreatment of breast, lung, digestive system, urogenital system,melanomas, sarcomas and neuroectodermic tumors, their metastases andrelapses, comprising the described antibodies and an appropriateexicipient.

In another representation of the present invention, the pharmaceuticalcompositions can be used for the localization and diagnosis in vivo ofbreast, lung, digestive system, urogenital system, melanomas, sarcomasand neuroectodrmico tumors, their metastases and relapses, comprisingthe described antibodies.

cDNA Synthesis and Gene Amplification by PCR (Polymerase chain reaction)of the Variable Region of MAb P3 and Mabai 1E10.

Cytoplasmic RNA was extracted from about 10⁶ hybridoma cells of P3(murine IgM MAb, that recognizes to the GM3 N-glycolylated ganglioside)or of 1E10 (antidiotype anti-P3 antibody). The RNA was extracted byusing the reagent TRIZOL (GIBCO BRL, Grand Island, N.Y.), according tothe manufacturer's instructions.

The cDNA synthesis reaction was carried out mixing 5 μg of the RNA, 25pmoles of Vh (complementary to the constant region of murine IgM forVHP3, and with the constant region of murine IgG1 for VH 1E10) or Vk(complementary to constant murine kappa region for both antibodies), 2.5mM of each dNTPs, 50 mM Tris-Hcl pH 7.5, 75 mM KCl, 10 mM DTT, 8 mMMgCl2 and 15 units of RNAse inhibitor in a 50 μl reaction mixture. Itwas heated at 70° C., for 10 minutes and slowly cooled up to 37° C.Then, 100 units of MLV reverse transcriptase enzyme were added and theincubation at 42° C. continued for one hour.

The variable regions VK and VH cDNAs were amplified by PCR. Shortly, 5μl cADN of VH or VK were mixed with 25 pmoles of specific primers, 2.5mM of each dNTP, 5 μl constituents of 10× buffer Taq DNA polymerase and1 unit of this enzyme. The samples were subjected to 25 thermal cyclesat 94.° C., 30 sec; 50° C., 30 sec; 72° C., 1 min, and a last incubationfor 5 minutes at 72° C.

Cloning and Sequencing of Amplified cDNA

The PCRs products of VH and VK (of the P3 and of the 1E10 respectively)were cloned into TA vector (TA Cloning kit. Promega, USA). The resultingclones were sequenced by the dideoxy method using T7 DNA Polymerase (T7sequencing kit, Pharmacia, Sweden).

Construction of Chimeric Genes

The VH VK genes were excised from TA vectors by enzymatic digestion andthey were cloned into the respective expression vectors (Coloma et al.(1992): Novel vectors for the expression of antibody molecules usingvariable regions generated by polymerase chain reaction, J. Immunol.Meth., 152: 89-104).

The VH genes were excised from the TA vector by enzymatic digestion withEcoRV and NheI and cloned in an expression vector (PAH 4604) that hasincluded a variable region human IgG1 and the histidinol resistancegene. The resultant constructions were P3VH-PAH4604 and 1E10VH-PAH4604.The VK genes were excised from TA vector by enzymatic digestion withEcoRV and SalI and cloned in an expression vector (PAG4622). This vectorhas included mycophenolic acid resistance gene and the human kappaconstant region. The resultant constructions were P3VK-PAG4622 and1E10VK-PAG4622.

Expression of Chimeric Antibodies Obtained from Mab P3 and Mabid 1E10.

NS-0 cells were electroporated with 10 μg of P3VK-PAG4622 or1E10VK-PAG4622, clones expressing human kappa light chains weretransfected with 10 μg of P3VH-PAH4604 or 1E1 OVH-PAH4604.

The DNAs were linearized by digestion with Pvul enzyme, precipitatedwith ethanol and dissolved in 50 μl of PBS. Approximately 10⁷ cells wereharvested by centrifugation and resupended in 0.5 ml of PBS togetherwith the digested DNA in an electroporation cuvette. After 10 minutes onice, the cells were given a pulse of 200 volts and 960 μF and left inice for a further 10 minutes. The cells were distributed into 96 wellsplate with D'MEM F12 plus 10% fetal calf serum. Two or four days later,it is added selective medium (D'MEM F12 with mycophenolic acid 0.45μg/mL or histidinol 10 mM, respectively). Transfected clones werevisible with the naked eyes 14 days later.

The presence of human antibody in the medium of the wells containingtransfected clones was measured by ELISA. Microtiter plate wells werecoated with goat anti-human kappa light chain (for human kappa chainproducing clones) or anti-human IgG (gamma chain specific) (for thecomplete antibody producing clones) antibodies. After washing with PBST(saline phosphate buffered solution containing 0.05% Tween 20), dilutedculture medium of the wells containing transfectants was added to eachmicrotiter well for one hour at 37° C. The wells were washed with PBS-Tand peroxidase of spicy radish-conjugated goat anti-human kappa lightchain or alkaline phosphatase-conjugated goat anti-human IgG (gammachain specific), were added and incubated at 37° C. one hour. The wellswere washed with PBS-T and substrate buffer containing o-phenylendiamineor p-nitrophenylphosphate, respectively, was added. After half hour,absorbance at 492 or 405 nm respectively, was measured.

Construction of the Humanized Antibodies P3hu and 1E10 hu byHumanization of T Cell Epitopes. Prediction of T Cell Epitopes

The sequences of P3 and 1E10 variable domains were analyzed with thealgorithm AMPHI (Margalit et al. (1987): Prediction of immunodominanthelper T cell antigenic sites from the primary sequence, J. Immunol.,138: 2213-2229). It searched helical amphipatic segments, with 7 or 11aminoacid residues, which have been associated with T immunogenicity.The program SOHHA also predicted helical hydrophobic segments. (Elliotet al. (1987). An hypothesis on the binding of an amphipatic, alphahelical sequence in li to the desotope of class II antigen, J. Immunol.,138: 2949-2952). Both algorithms predicted which segments from variableregion sequences of antibodies P3 and 1E10 could be presented toT-helper cells in the context of MHC class 11 molecules.

Homology Analysis with Human Immunoglobulins.

The amino acid sequences of murine variable regions were compared withthe immunoglobulin sequences included in the GeneBank and EMBL database(available in Internet). The most homologous human variable regionsequence was determined for each antibody. Software PC-TWO HIBIO PROSIS06-00 (Hitachi) was used for sequences homology searching.

Analysis for the Immunogenicity Reduction.

The aim of the method is to reduce immunogenicity breaking or humanizingpotential immunogenic T epitopes, with a minimum of changes. The methodcomprises judiciously replacement of few amino acid residues, locatedinto helical amphipatic segments. The amino acids, which are mainlyresponsible for canonical structures and also the residues in theimmediate neighbourhood of the CDRs or into Vernier zone, must beretained.

According to the method, murine variable region sequences were comparedwith the most homologous human sequence and different aminoacid residuesat each position between the murine MAb and the most homologous humansequence were identified, only residues into FRs were taken into account(Kabat (1991), Sequences of proteins of immunological interest, FifthEdition, National Institute of Health), the previously defined residueswere replaced by those residues present in the most homologous humansequence. Replacements were made by directed mutagenesis techniques.

Residues involved in three-dimensional structure of the binding sitewere not mutated; it could affect antigen recognition. Additionalinformation about the influence of the replacements in the tertiarystructure can be obtained by molecular modelling of the antigen bindingsite.

The presence of proline residues into the helical amphipatic segment andthe fact that a certain murine residues don't appear in the sameposition in the most homologous human sequence but be frequent in otherhuman immunoglobulins, must be kept in mind. For that reason there isnot a unique ensemble of murine amino acids to be replaced into theframeworks. It is possible to obtain different versions of the modifiedantibody with different numbers of replacements. The mutations werecarried out by over-lapping of PCRs.

Cloning and Expressing Humanized Antibodies P3hu and 1E10hu.

The genetic constructions corresponding to the P3hu and 1E10hu, werecloned in expression vectors following the method described for thechimeric antibodies. The resultants constructions were P3VKhu-PAG4622 or1E10Vkhu-PAG4622 and P3VHhu-PAH4604 and 1E10VHhu-PAH4604. They weretransfected into NS-0 cells following the protocol described previouslyfor chimeric antibodies.

Purification of the Recombinant Antibodies.

The recombinant antibodies were purified by affinity chromatographyusing protein A (Pharmacia, Upssala, Sweden).

Biological Activity.

The specific binding to antigen measured by ELISA tested the biologicalactivity of the recombinant antibodies.

For recombinant MAb P3, microtiter plates were coated with GM3(NeuGc)ganglioside in methanol. After drying one hour, unspecific binding wasblockade with bovine sera albumin (BSA) 1% in Tris-HCl buffer, incubatedfor one hour at 37° C. The wells were washed with PBS and incubated for1 hour at 37° C. with purified recombinant Mab P3. The wells were washedwith tris-HCl and a goat anti-human antibody conjugated with alkalinephosphatase was added and incubated at 37° C. for one hour. Finally, thewells were washed and the substrate buffer containingp-nitrophenylphosphate was added. After half hour absorbance at 405 or492 nm respectively, was measured.

For recombinant Mabai 1E10, the ELISA assay was similar, except thatwells were coated with Mab P3 and washing were made with PBS-0.05% Tween20.

EXAMPLES

In the following examples all the enzymes used, as well as reagents andmaterials were obtained from commercial sources unless the opposite isspecified.

Example 1 Obtaining of Chimeric MAb P3

The cDNA synthesis was obtained by a reaction with reverse transcriptaseenzyme, starting with RNA from the hybridoma producing Mab P3, asdescribed previously. The sequence of the specific primers used in thisreaction is shown:

For VH: 5′ AGGTCTAGAA(CT)CTCCACACAC AGG(AG)(AG)CCAGTGGATA GAC 3′ For VK:5′ GCGTCTAGAACTGGATGGTGGGAAGATGG 3′cDNA VHP3 and cDNA VKP3 were amplified by PCR using Taq Polymerase andspecific primers. The restriction sites included in the primers wereECORV/NHEI, for VH and ECORV/SALI for VK. The primers sequences usedwere the following:

For VH: Primer 1 (signal peptide): 5′GGGGATATCCACCATGG(AG)ATG(CG)AGCTG(TG)GT(CA)AT (CG)CTCTT 3′ Primer 2 (CH1):5′ GGGGCTAGCTGCAGAGACAGTGACCAGAGT 3′ For VK: Primer 1 (signal peptide):5′ GGGGATATCCACCATGGAG(TA)CAC A(GT)(TA)CTCAGGTCTT T(GA)T 3′ Primer 2(Ck): 5′ AGCGTCGACTTACGTTT(TG)ATTTCCA(GA)CTT(GT)GTCCC 3′

PCR products were cloned into TA vector (TA cloning kit, Invitrogen).Twelve independent clones were sequenced by the dideoxy method using T7DNA Pol (Pharmacia). By homology search analysis it was determined themost homologous sequence group for VHP3 and VKP3. VHP3 and VKP3sequences (FIGS. 1 and 2) have high homology with groups IB and Vrespectively according to Kabat's classification.

After digestion with the restriction enzymes ECORV and NHEI for VHP3 andwith ECORV and SALI for VKP3, they were cloned in the expression vectorspreviously digested with the same enzymes, PAH4604 and PAG4622 for VHand VK respectively. These expression vectors were donated by SherieMorrison (UCLA, Calif., USA), they are suitable for immunoglobulinsexpression in mammalian cells. The vector PAH 4604 have included thehuman constant region IgG1 and the PAG 4622 human (Coloma et al. (1992):Novel vectors for the expression of antibody molecules using variableregions generated by polymerase chain reaction, J. Immunol. Meth., 152:89-104). The resultant constructs were P3VH-PAH4604 and P3VK-PAG4622.

NS-0 cells were transfected with 10 .μg of P3VK-PAG4622, a cloneexpressing light chain was transfected with 10 .μg P3VH-PAH4604, in bothcases DNA is linearized with Pvul, ethanol precipitated and dissolved in50 μl of PBS before transfection.

Approximately 10⁷ cells were harvested by centrifugation and resupendedin 0.5 ml of PBS together with the digested DNA in an electroporationcuvelte. After 10 minutes on ice, the cells were given a pulse of 200volts and 960 μF. and left in ice for a further 10 minutes. The cellswere distributed into 96 wells plate with D'MEM F12 plus 10% fetal calfserum. Two or four days later, it is added selective medium (D'MEM F12with mycophenolic acid 0.45 .μg/mL or histidinol 10 mM, respectively).Transfected clones were visible with the naked eyes 14 days later.

The presence of human antibody in the medium of wells containingtransfected clones was measured by ELISA. Microtiter plate wells werecoated with goat anti-human kappa light chain (for human kappa chainproducing clones) or anti-human IgG (gamma chain specific) (for thecomplete antibody producing clones) antibodies. After washing with PBST(saline phosphate buffered solution containing 0.05% Tween 20), dilutedculture medium of the wells containing transfectants was added to eachMicrotiter well for one hour at 37°.C. The wells were washed with PBS-Tand peroxidase of spicy radish-conjugated goat anti-human kappa lightchain or alkaline phosphatase-conjugated goat anti-human IgG (gammachain specific), were added and incubated at room temperature one hour.The wells were washed with PBS-T and substrate buffer containingo-phenylendiamine or p-nitrophenylphosphate, respectively, was added.After half hour absorbance at 492 or 405 nm respectively, was measured.

Example 2 Obtaining Different Versions of the Humanized Antibody P3

Murine VHP3 and VKP3 sequences (FIGS. 1 and 2) were compared with humansequences. FIGS. 3 and 4 show the most homologous human sequences.Helical amphipatic regions or potential T cell epitopes were searched onmurine P3 variable region sequences and according with the method ajudiciously strategy for aminoacid replacements was established in orderto break or humanize potential T cell epitopes into the murinesequences.

The analysis on VHP3 rendered (FIG. 3) 2 amphipatic segments, the firstone embraces CDR1, FR2 and some residues of the CDR2, the second oneembraces the end of FR3 and CDR3. The main differences of murinesequence in comparison with the most homologous human sequence werefounded in CDRs or residues involved with the three dimensionalstructure of the binding site. For that reason it was decided do notreplace any aminoacid in murine VHP3.

The analysis for VKP3 rendered also 2 amphipatic segments (FIG. 4), thefirst segment embraces FR1, the second one embraces CDR2 and someresidues of the FR3. It was decide to replace residues at positions 8,9, 10, 11 and 13 by residues at the same position in the most homologoushuman sequence. The amino acids aminoacidos His, Lys, Phe, Met and Thrwere replaced by Pro, Ser, Ser, Leu, and Ala, respectively. Thereplacements were made by PCR overlapping (Kammann et al. (1989) Rapidinsertional mutagenesis of DNA by polymerase chain reaction (PCR),Nucleic Acids Res., 17: 5404) using primers 1 and 2 and 3 and 4 whosesequences are the following:

Primer 1: 5′ ATGACCCAGTCTCCTTCTTCTCTTTCCGCGTCAGTAG GAGAC 3′ Primer 2:5′ AGCGTCGACTTACGTTT(TG)ATTTCCA(GA)CT- T(GT)GTCCC 3′ Primer 3:5′ GTCTCCTACTGACGCGGAAAGAGA-AGAAGGAGACTG GGTCAT 3′ Primer 4:5′GGGGATATCCACCATGGAG(TA)CACA(GT)(TA)CTC AGGTCTTT (GA)T 3′

The point mutations were verified by sequencing. The resultant constructwas P3Vkhu and it was cloned in PAG 4622 expression vector. Theresultant construct was P3VKhu-PAG4622.

To express the humanized antibody P3, NS-0 cells were transfected withP3VH-PAH4604 and P3VKhu-PAG4622 P3hu antibody was transfected followingthe same procedure of electroporation and detection described previouslyfor the chimeric antibodies.

Example 3 Biological Activity of Chimeric MAb P3

The specific binding to antigen measured by ELISA tested the biologicalactivity of the Mab P3 chimeric.

For recombinant MAb P3, microtiter plates were coated with GM3(NeuGc)ganglioside in methanol. After drying one hour at 37° C., unspecificbinding was blockade with bovine sera albumin (BSA) 1% in Tris-HClbuffer, incubated for one hour at 37° C. The wells were washed with PBSand incubated for 1 hour at 37° C. with purified recombinant Mab P3. Thewells were washed with tris-HCl and a goat anti-human antibodyconjugated with alkaline phosphatase was added and incubated at 37° C.for one hour. Finally, the wells were washed with Tris-HCl and thesubstrate buffer containing p-nitrophenylphosphate was added. After halfhour absorbance at 405 nm, was measured.

Mab Ti chimeric was used as negative control.

FIG. 5 shows the specific binding of Mab P3 chimeric to the antigen.

Example 4 Obtaining of Chimeric MAb 1E10

The cDNA synthesis was obtained by a reaction with reverse transcriptaseenzyme, starting with RNA from the hybridoma producing Mab 1E10, asdescribed previously. The sequence of the specific primers used in thisreaction is shown following:

For VH: 5′GGGGCTAGCTGAGGAGACTGTGAGAGTGGT 3′ For VK:5′GCGTCTAGAACTGGATGGTGGGAAGATGGA 3′cDNA VH1E10 and cDNA VK1E10 were amplified by PCR using Taq Pol andspecific primers.

For VH: Primer 1 (signal peptide): 5′GGGGATATCCACCATGG(AG)ATG-(CG)AGCTG(TG)GT(CA)AT(CG)CTCTT 3′ Primer 2 (CH1):5′ GGGGCTAGCTGAGGAGACTGTGAGAGTGG T 3′ For VK: Primer 1 (signal peptide):5′GGGGTTAACCACCATGAGG (GT)C-CCC(AT)GCTCAG(CT)T(CT)CT(TG)GG(GA)3′ Primer2 (Ck): 5′AGCGTCGACTTACGTTT(TG)ATTTCCA(GA)C TT(GT)GTCCC3′

PCR products were cloned into TA vector (TA cloning kit, Invitrogen).Twelve independent clones were sequenced (FIGS. 7 and 8) by the dideoxymethod using T7 DNA Pol (Pharmacia). By homology search analysis it wasdetermined the most homologous sequence group for VHIE10 and VK1E10.VH1E10 and VK1E10 sequences have high homology with groups miscellaneousand V respectively according to Kabat's classification.

After digestion with the restriction enzymes ECORV and NHEI for VH1E10and with HincII and SALI for VKIE10, they were cloned in the expressionvectors previously digested with appropriated enzymes, PAH4604 andPAG4622 for VH and VK respectively. These expression vectors weredonated by Sherie Morrison (UCLA, Calif., USA), they are suitable forimmunoglobulins expression in mammalian cells. The vector PAH 4604 haveincluded the human constant region IgG1 and the PAG 4622 human (Colomaet al. (1992): Novel vectors for the expression of antibody moleculesusing variable regions generated by polymerase chain reaction, J.Immunol. Meth., 152: 89-104). The resultant constructs 1E10VH-PAH4604and 1E10VK-PAG4622.

NS-0 cells were transfected with 10 μg of 1E10VK-PAG4622, a cloneexpressing light chain was transfected with 10 μg 1E10VH-PAH4604, inboth cases DNA is linearized with Pvul, ethanol precipitated anddissolved in 50 μl of PBS before transfection.

Approximately 10⁷ cells were harvested by centrifugation and resupendedin 0.5 ml of PBS together with the digested DNA in an electroporationcuvette. After 10 minutes on ice, the cells were given a pulse of 200volts and 960 .μF. and left in ice for a further 10 minutes. The cellswere distributed into 96 wells plate with D'MEM F12 plus 10% fetal calfserum. Two or four days later, it is added selective medium (D'MEM F12with mycophenolic acid 0.45 μg/mL or histidinol 10 mM, respectively).Transfected clones were visible with the naked eyes 14 days later.

The presence of human antibody in the medium of wells containingtransfected clones was measured by ELISA. Microtiter plate wells werecoated with goat anti-human kappa light chain (for human kappa chainproducing clones) or anti-human IgG (gamma chain specific) (for thecomplete antibody producing clones) antibodies. After washing with PBST(phosphate buffered saline containing 0.05% Tween 20), diluted culturemedium of the wells containing transfectants was added to eachMicrotiter well for one hour at 37° C. The wells were washed with PBS-Tand peroxidase of spicy radish-conjugated goat anti-human kappa lightchain or alkaline phosphatase-conjugated goat anti-human IgG (gammachain specific), were added and incubated at room temperature one hour.The wells were washed with PBS-T and substrate buffer containingo-phenylendiamine or p-nitrophenylphosphate, respectively, was added.After half hour absorbance at 492 or 405 nm respectively, was measured.

Example 5 Obtaining Different Versions of the Humanized Antibody 1E10

Murine VH1E10 VK1E10 sequences (FIGS. 6 and 7) were compared with humansequences, FIGS. 8 and 9 shown the most homologous human sequences.Helical amphipatic regions or potential T cell epitopes were searched onmurine 1E10 variable region sequences and according with the method ajudiciously strategy for aminoacid replacements was established in orderto break or humanize potential T cell epitopes into the murine sequences

The analysis on VH1E10 rendered (FIG. 8) 3 amphipatic segments, thefirst one embraces FR1, the second one embraces FR2, the third oneembraces FR3. It was decide to replace residues at positions 5, 40, 42and 87 (83 according to Kabat's numbering) by residues at the sameposition in the most homologous human sequence. The amino acids Gln,Arg, Glu and they were replaced by Val, Ala, Gly and Arg, respectively.

The replacements were made by PCR overlapping (Kammann et al. (1989)Rapid insertional mutagenesis of DNA by polymerase chain reaction (PCR),Nucleic Acids Res., 17: 5404) using different set of primers.

Primers for mutation at position 5 of the heavy chain were 1 and 2 and 3and 4 whose sequences are the following:

Primer 1: 5′ CAGGTTCAGCTGGTGCAGTCTGGAGCT 3′ Primer 2:5′ GGGGCTAGCTGAGGAGACTGTGAGAGTGGT 3′ Primer 3:5′ AGCTCCAGACTGCACCAGCTGAACCTG 3′ Primer 4:5′GGGGATATCCACCATGG(AG)ATG(CG)AGCTG (TG)GT(CA)AT(CG)CTCTT 3′

After checking by sequence the point mutation at position 5, mutationsat positions 40 and 42 were introduced.

Primer for mutations at positions 40 and 42 of the heavy chain:

Primer 1: 5′ TGGGTGAGGCAGGCGCCTGGGCAGGGACTTGAG 3′ Primer 2:5′ GGGGCTAGCTGAGGAGACTGTGAGAGTGGT 3′ Primer 3:5′ CTCAAGTCCCTGCCCAGGCGCCTGCCTCACCCA 3′ Primer 4:5′GGGGATATCCACCATGG(AG)ATG(CG)AGCTG(TG) GT(CA)AT(CG)CTCTT 3′

After checking by sequence the point mutation at positions 40 and 42,mutation at positions 87 (83 according to Kabat's numbering) wasintroduced.

Primer for mutations at position 87 (83 according to Kabat's numbering)of the heavy chain:

Primer 1: 5′ CTCAGCAGGCTGCGGTCTGAGGACTCT 3′ Primer 2:5′ GGGGCTAGCTGAGGAGACTGTGAGAGTGGT 3′ Primer 3:5′ AGAGTCCTCAGACCGCAGCCTGCTGAG 3′ Primer 4:5′GGGGATATCCACCATGG(AG)ATG(CG)AGCTG (TG)GT(CA)AT(CG)CTCTT 3′

Other replacements were not made because residues were involved in thethree dimensional structure of the binding site.

The point mutations were verified by sequencing. The resultant constructwas 1E10VHhu and it was cloned in PAH4604 expression vector. Theresultant construct was 1E10VH-PAH4604.

The analysis for VKIE10 rendered also 3 amphipatic segments (FIG. 9),the first segment embraces FR1, the second one embraces CDR1 and thethirst one embraces FR3. It was decide to replace residues at positions7, 8 and 15 by residues at the same position in the most homologoushuman sequence. The amino acids Thr, Thr and Leu were replaced by Ser,Pro and Val, respectively. The replacements were made by PCR overlapping(Kammann et al. (1989) Rapid insertional mutagenesis of DNA bypolymerase chain reaction (PCR), Nucleic Acids Res., 17: 5404) usingprimers 1 and 2 and 3 and 4 whose sequences are the following:

Primers for mutation at positions 7, 8 and 15 of the light chain:

Primer 1: 5′CAGATGACACAGTCTCCTTCCTCCCTGTCTGCCTCTGT GGGAGA-CAGAGTC 3′Primer 2: 5′AGCGTCGACTTACGTTT(TG)ATTTCCA(GA)CTT (GT)GTCCC 3′ Primer 3:5′GACTCTGTCTCCCACAGAGGCAGACAGGGAGGAAGGAG ACTGTGTCATCTG 3′ Primer 4:5′GGGGTTAACCACCATGAGG(GT)CCCC(-AT)GCTCA G(CT)T(CT)CT(TG)GG(GA) 3′

The point mutations were verified by sequencing. The resultant constructwas 1 EI OVkhu and it was cloned in PAG 4622 expression vector. Theresultant construct was 1E10 VKhu-PAG4622.

To express the humanized antibody 1E10, NS-0 cells were transfected with1E10 VHhu-PAH4604 and 1 EI OVKhu-PAG4622 1E10 hu antibody wastransfected following the same procedure of electroporation anddetection described previously for the chimeric antibodies.

Example 6 Biological Activity of Chimeric MAb1E10

The specific binding to antigen measured by ELISA tested the biologicalactivity of the Mab 1E10 chimeric.

For recombinant MAb 1 E10, Microtiter plates were coated with Mab P3.After washing with PBST (saline phosphate buffered solution containing0.05% Tween 20), unspecific binding was blockade with bovine seraalbumin (BSA) 1% in PBST, incubated for one hour at 37° C.

The wells were washed and incubated for 1 hour at 37° C. with purifiedrecombinant Mab 1E10. The wells were washed with PBST and a goatanti-human antibody conjugated with alkaline phosphatase was added andincubated at 37° C. for one hour. Finally, the wells were washed withPBST and the substrate buffer containing p-nitrophenylphosphate wasadded. After half an hour, the absorbance at 405 nm respectively, wasmeasured. Mab C5 chimeric was used as negative control.

FIG. 10 shows the specific binding of Mab 1E10 chimeric to Mab P3.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: VHP3 DNA and deduced amino acid sequences. Sequences are alignedaccording Kabat's numbering (Kabat et al. (1991), Sequences of proteinsof immunological interest, Fifth Edition, National Institute of Health),CDRs appeared marked with dotted lines.

FIG. 2: VKP3 DNA and deduced amino acid sequences. Sequences are alignedaccording Kabat's numbering (Kabat and collaborators (1991), Sequencesof proteins of immunological interest, Fifth Edition, National Instituteof Health), CDRs appeared marked with dotted lines.

FIG. 3: VHP3 was aligned with the most homologous human sequence.Amphipatic segments are underlined and CDRs in bold.

FIG. 4: VKP3 was aligned with the most homologous human sequence.Amphipatic segments are underlined and CDRs in bold.

FIG. 5: Specific binding to GM3(NeuGc) by chimeric Mab P3. Differentconcentrations of Mab P3 and MAb TI (negative control) were tested byELISA. Microtiter plates were coated with GM3(NeuGc) and GM3(NeuAc)(negative control) ganglioside in methanol and specific binding wasmeasured.

FIG. 6: VH1E10 DNA and deduced amino acid sequences. Sequences arealigned according Kabat's numbering (Kabat and collaborators (1991),Sequences of proteins of immunological interest, Fifth Edition, NationalInstitute of Health), CDRs appeared marked with dotted lines.

FIG. 7: VK1E10 DNA and deduced amino acid sequences. Sequences arealigned according Kabat's numbering (Kabat et al. (1991), Sequences ofproteins of immunological interest, Fifth Edition, National Institute ofHealth), CDRs appeared marked with dotted lines.

FIG. 8: VH1E10 was aligned with the most homologous human sequence.Amphipatic segments are underlined and CDRs in bold.

FIG. 9: VK1E10 was aligned with the most homologous human sequence.Amphipatic segments are underlined and CDRs in bold.

FIG. 10: Specific binding murine Mab P3 by chimeric Mab 1E10. Differentconcentrations of Mab 1E10 and MAb C5 (negative control) were tested byELISA. Microtiter plates were coated with Mab P3 and Mab A3 (negativecontrol) and specific binding was measured.

1. A chimeric monoclonal antibody derived from the murine anti-idiotypicmonoclonal antibody 1E10 that recognizes Mab P3 and produced by thehybridoma cell line with deposit number ECACC 97112901, wherein thehypervariable domains of its heavy and light chains comprise thefollowing sequences: HEAVY CHAIN (SEQ ID NO: 15) CDR1: SYDIN; (SEQ IDNO: 16) CDR2: WIFPGDGSTKYNEKFKG; (SEQ ID NO: 17) CDR3: EDYYDNSYYFDY;LIGHT CHAIN (SEQ ID NO: 18) CDR1: RASQDISNYLN; (SEQ ID NO: 19) CDR2:YTSRLHSG; (SEQ ID NO: 20) CDR3: QQGNTLPWT.


2. The monoclonal antibody of claim 1, wherein the framework regions(FRs) of its heavy and light chains comprise the following sequences:HEAVY CHAIN (SEQ ID NO: 21) FR1: QVQLQQSGAELVKPGASVKLSCKASGYTFT; (SEQ IDNO: 22) FR2: WVRQRPEQGLEWIG; (SEQ ID NO: 23) FR3:KATLTTDKSSSTAYMQLSRLTSEDSAVYFCAR; (SEQ ID NO: 24) FR4: WGQGTTLTV; LIGHTCHAIN (SEQ ID NO: 25) FR1: DIQMTQTTSSLSASLGDRVTISC; (SEQ ID NO: 26) FR2:WYQQKPDGTVKLLIY; (SEQ ID NO: 27) FR3: VPSRFSGSGSGTDYSLTISNLEQEDIATYFC;(SEQ ID NO: 28) FR4: FGGGTKLESK.


3. The monoclonal antibody of claim 1, wherein for its humanization andpreserving the binding properties to the antigen, the monoclonalantibody further comprises at least one of the following substitutions:LIGHT CHAIN: Position 7: Thr by Ser; Position 8: Thr by Pro; Position15: Leu by Val; HEAVY CHAIN Position 5: Gln by Val; Position 40: Arg byAla; Position 42: Glu by Gly; Position 87 (83 according to Kabat'snumbering): Thr by Arg.


4. The monoclonal antibody of claim 1, wherein the constant region ofthe heavy chain comprises the amino acid sequence of gamma-1 chain, andthe constant region of the light chain comprises the amino acid sequenceof a kappa chain, both being derived from human immunoglobulins.
 5. Acell line that produces any of the monoclonal antibodies of claim
 1. 6.A pharmaceutical composition for the treatment of malignant breasttumors and melanomas, their metastases and relapses, said compositioncomprises the monoclonal antibodies of claim
 1. 7. A pharmaceuticalcomposition for in vivo localization and identification of malignantbreast tumors and melanomas, their metastases and relapses, saidcomposition comprises the monoclonal antibodies of claims which comprisethe monoclonal antibodies of claim
 1. 8. The use of a monoclonalantibody of claim 1 for the manufacture of a medicament useful for thetreatment of malignant tumors of breast and melanomas, their metastasesand relapses.
 9. A method for the treatment of breast tumors andmelanomas in a mammal In need of the treatment, said method comprisesadministrating an effective dosage of a chimeric monoclonal antibody,said chimeric monoclonal antibody comprises a chimeric monoclonalantibody derived from the murine anti-idiotypic monoclonal antibody 1E10that recognizes Mab P3 and produced by the hybridoma cell line withdeposit number ECACC 97112901, wherein the hypervariable domains of itsheavy and light chains comprise the following sequences: HEAVY CHAIN(SEQ ID NO: 15) CDR1: SYDIN; (SEQ ID NO: 16) CDR2: WIFPGDGSTKYNEKFKG;(SEQ ID NO: 17) CDR3: EDYYDNSYYFDY; LIGHT CHAIN (SEQ ID NO: 18) CDR1:RASQDISNYLN; (SEQ ID NO: 19) CDR2: YTSRLHSG; (SEQ ID NO: 20) CDR3:QQGNTLPWT.


10. The method of claim 9, wherein the framework regions (FRs) of itsheavy and light chains comprise the following sequences: HEAVY CHAIN(SEQ ID NO: 21) FR1: QVQLQQSGAELVKPGASVKLSCKASGYTFT; (SEQ ID NO: 22)FR2: WVRQRPEQGLEWIG; (SEQ ID NO: 23) FR3:KATLTTDKSSSTAYMQLSRLTSEDSAVYFCAR; (SEQ ID NO: 24) FR4: WGQGTTLTV; LIGHTCHAIN (SEQ ID NO: 25) FR1: DIQMTQTTSSLSASLGDRVTISC; (SEQ ID NO: 26) FR2:WYQQKPDGTVKLLIY; (SEQ ID NO: 27) FR3: VPSRFSGSGSGTDYSLTISNLEQEDIATYFC;(SEQ ID NO: 28) FR4: FGGGTKLESK.


11. The method of claim 9, wherein for its humanization and preservingthe binding properties to the antigen, it includes at least one of thefollowing substitutions: LIGHT CHAIN: Position 7: Thr by Ser; Position8: Thr by Pro; Position 15: Leu by Val; HEAVY CHAIN Position 5: Gln byVal; Position 40: Arg by Ala; Position 42: Glu by Gly; Position 87 (83according to Kabat's numbering): Thr by Arg.


12. A method for the in vivo localization and identification ofmalignant breast tumors and melanomas to a mammal in need of thelocalization and identification, said method comprises administering achimeric monoclonal antibody comprising a chimeric monoclonal antibodyderived from the murine anti-idiotypic monoclonal antibody 1E10 thatrecognizes Mab P3 and produced by the hybridoma cell line with depositnumber ECACC 97112901, wherein the hypervariable domains of its heavyand light chains comprise the following sequences: HEAVY CHAIN (SEQ IDNO: 15) CDR1: SYDIN; (SEQ ID NO: 16) CDR2: WIFPGDGSTKYNEKFKG; (SEQ IDNO: 17) CDR3: EDYYDNSYYFDY; LIGHT CHAIN (SEQ ID NO: 18) CDR1:RASQDISNYLN; (SEQ ID NO: 19) CDR2: YTSRLHSG; (SEQ ID NO: 20) CDR3:QQGNTLPWT.


13. The method of claim 12, wherein the mammal is a human.